TECO® Cyprinid Vitellogenin ELISA

The TECO® Cyprinid Vitellogenin ELISA kit is a sensitive sandwich enzyme linked immunosorbent assay for the quantitative determination of vitellogenin in serum, WBH and mucus of cyprinids.

Vitellogenin determination is one of the core endpoints in screening and testing for endocrine disrupting chemicals standardized in the OECD Guidlines for the testing of chemicals for estrogenic activity:

OECD (2009), Test No.229

OECD (2009),Test No.230

OECD (2011), Test No.234

Vitellogenin determination is used in ecotoxicological studies to determine estrogenic compounds in the water.

Vitellogenin levels can be used to determine the sex and the sexual maturation of fish.

For mucus samples: Extraction Buffer and validated Sampling Swabs are not part of this kit. Please order TECO® Mucus Collection Set (TE1034) separately.

Assay Principle

The TECO® Cyprinid Vitellogenin EIA Kit is a 96 well immuno-capture ELISA product. Serum, WBH and mucus samples are incubated with the vitellogenin specific antibody coated microtiter plate. After unbound material is washed out, a polyclonal biotinylated antibody binds to the vitellogenin. In the following incubation step, a streptavidin-peroxidase conjugate binds to the biotinylated antibody. In the final substrate reaction, the color development is directly proportional to the amount of vitellogenin in the sample.

Assay Procedure

All determinations (standards, controls and samples) should be assayed in duplicate. When performing the assay, the standards, controls and samples should be pipetted as fast as possible (<15 minutes).

To avoid distortions due to differences in incubation times, HRP conjugate, substrate solution and stop solution should be added to the plate in the same order and with the same time interval as the samples. A multichannel pipette is essential.

Allow all reagents to stand at room temperature (20–25 °C) for at least 30 minutes. During all incubation steps, plates should be sealed with the adhesive foil or a plastic cover. For light protection, incubate in a dark chamber or cover plate with aluminum foil.

Allocate the wells of the microtiterplate 1 for standards, controls and samples.

Measure the color reaction within 10 minutes at 450 nm (reference filter between 590–650 nm). If the extinction of the standard A (35 ng/ml) exceeds 3.0, the measurement may be repeated at 405 nm.

Fish

Cyprinid

Carp (Caprinus carpio)

Goldfish (Carassius auratus)

Zebrafish (Danio rerio)

Medaka, Japanese rice fish (Oryzias latipes)

Fathead Minnow (Pimephales promelas)

In oviparous animals, vitellogenin (VTG) is an estrogen induced yolk precursor protein mainly synthesized in the liver to be deposited in the maturing oocytes, where it is split in the yolk proteins lipovitellin 1, lipovitellin 2 and phosvitin. These yolk proteins serve as nourishment storage for the developing embryos. Non-physiological induction of vitellogenin in males or in juvenile fish is thought to indicate an estrogen mediated endocrine disruption. Therefore VTG determination is one of the core endpoints in screening and testing for endocrine disrupting chemicals standardized in the OECD Guidelines for the testing of chemicals for estrogenic activity. Normally vitellogenin is measured in blood samples or whole body homogenate (WBH) – both sample types require invasive and destructive treatment of the fish. Blood is difficult to collect, in particular where very small fish are concerned, or in approaches where the animals must survive sampling. This is particularly important in field monitoring in order to avoid impact on the population under investigation. Recently several cell types have been shown to produce VTG after estrogen stimulation, including those of the epidermal mucosa. Even though the VTG concentration in the skin mucus is an order of magnitude lower than in blood serum or in body homogenates (containing liver tissue), the skin mucosa is very well suited as a matrix to determine exogenous VTG induction caused by environmental chemicals with affinity to estrogen receptors. By using a highly sensitive ELISA in combination with an unique sampling and extraction system the determination of mucosa born VTG determination has the following advantages:

Non-destructive and thereby allowing several subsequent samplings in order to record a kinetic of VTG induction with a maximum known to appear after 7 days of exposure. Therefor Mucosa test are fully compatible with acute as well as chronical OECD test methods.